Cyclic peptides that bind to urokinase-type plasminogen activator receptor

ABSTRACT

Cyclic peptide compounds having 11 amino acids joined by a linking unit L, such that the linear dimension between the C.sup.α  carbon of the first amino acid and the C.sup.α  carbon of eleventh amino acid is between about 4 and 12 Ångstrom units; are useful for inhibiting the binding of uPA to the uPAR receptor. Methods for using the cyclic peptide compounds, and compositions containing them, for inhibiting the growth or metastasis of cancerous tumors are also disclosed.

TECHNICAL FIELD

The present invention relates to certain cyclic peptides that bind tothe cell surface receptor for urokinase-type plasminogen activator and,thus, are capable of inhibiting the binding of urokinase-typeplasminogen activator to this cell surface receptor. The invention alsorelates to pharmaceutical compositions containing these peptides and tothe use of these peptides to inhibit the binding of urokinase-typeplasminogen activator to its cell surface receptor. Effects derived fromthe inhibition of binding of urokinase-type plasminogen activator to itscell surface receptor include the inhibition of proteolysis; theinhibition of programmed gene expression; the inhibition of cellmotility, migration, and morphogenesis; the inhibition of the activationof certain pro-growth factors to the active form of the growth factor;the inhibition of angiogenesis; the inhibition of tumor metastasis; theinhibition of retinal neovascularization in the treatment of certainforms of blindness; and the inhibition of tissue remodeling as atreatment for inflammatory diseases, such as arthritis. The peptides ofthe invention that are capable of carrying a suitable radioactive,fluorogenic, chromogenic, or chemical label can also be used toquantitate urokinase-type plasminogen activator receptor levels intissue samples and can be used, therefore, as diagnostic and prognostictools in all diseases where the receptor plays a pathological role,including those mentioned above.

BACKGROUND OF THE INVENTION

Urokinase-type plasminogen activator (uPA) has been identified as theinitiator of a major amplified cascade of extracellular proteolysis.This cascade, when regulated, is vital to certain normal physiologicalprocesses but, when dysregulated, is strongly linked to pathologicalprocesses, such as cell invasion and metastasis in cancer. Dano/ et al.,Adv. Cancer Res., 44:139-266 (1985). Cells express uPA as an inactiveform, pro-uPA or single-chain uPA, which then binds to its receptor,uPAR. This binding event is necessary for activation to two-chain uPA.Ellis et al., J. Biol. Chem., 264:2185-88 (1989). The amino acidsequence for human pro-uPA is as follows (SEQ ID NO:1):

    Ser Asn Glu Leu His Gln Val Pro Ser Asn Cys Asp Cys Leu Asn Gly               1                                   10                                        Gly Thr Cys Val Ser Asn Lys Tyr Phe Ser Asn Ile His Trp Cys Asn Cys                       20                                      30                        Pro Lys Lys Phe Gly Gly Gln His Cys Glu Ile                                                           40                                                

The sequence of amino acids of pro-uPA are represented above by theirstandard three-letter abbreviations as follows:

    ______________________________________                                        Amino Acid         Three-letter Symbol                                        ______________________________________                                        Alanine            Ala                                                        Arginine           Arg                                                        Asparagine         Asn                                                        Aspartic acid      Asp                                                        Cysteine           Cys                                                        Glutamine          Gln                                                        Glutamic acid      Glu                                                        Glycine            Gly                                                        Histidine          His                                                        Isoleucine         Ile                                                        Leucine            Leu                                                        Lysine             Lys                                                        Methionine         Met                                                        Phenylalanine      Phe                                                        Proline            Pro                                                        Serine             Ser                                                        Threonine          Thr                                                        Tryptophan         Trp                                                        Tyrosine           Tyr                                                        Valine             Val                                                        ______________________________________                                    

The structure of pro-uPA is shown in FIG. 1.

uPA is a three-domain protein comprising (1) an N-terminal epidermalgrowth factor-like domain, (2) a kringle domain, and (3) a C-terminalserine protease domain. The receptor for pro-uPA (uPAR) is amulti-domain protein anchored by a glycolipid to the cell membrane, thusensuring that activation of uPA is a pericellular event. Behrendt etal., Biol. Chem. Hoppe-Seyler, 376:269-79 (1995). uPA activity isconfined to the cell surface by plasminogen activator inhibitors (PAI-1and PAI-2), which bind to and inactivate the bound uPA. This tightcontrol of uPA activity is necessary because uPA acts upon a substrate,plasminogen, that is present at a high concentration in plasma. Robbins,Meth. Enzymol., 19:184-99 (1970). The product of uPA's action uponplasminogen, plasmin, is a powerful broad spectrum protease that notonly degrades extracellular matrix proteins directly, but also activatesthe latent forms of other proteases, including several metalloproteases.Werb et al., N. Eng. J. Med., 296:1017-1023 (1977); Mignatti et al.,Cell, 47:487-98 (1986); He et al., Proc. Natl. Acad. Sci. USA,86:2632-36 (1989); and Matrisian, Bioessays, 14:455-63 (1992).

In tumor biology, the link between extracellular proteolysis andangiogenesis is clearly evident. Break-up and dissolution of existingextracellular matrix is necessary in order to create new space for bloodvessels to grow into. The processes of proteolysis and angiogenesis arehighly coordinated. For example, two pre-eminent angiogenic growthfactors, basic fibroblast growth factor and vascular endothelial growthfactor markedly up-regulate the production of uPA. (Montesano et al.,Proc. Natl. Acad. Sci. USA, 83:7297-7301 (1986); Pepper et al., Biochem.Biophys. Res. Comm., 181:902-906 (1991)) and the expression of uPAR byendothelial cells (Mignatti et al., J. Cell Biol., 113:1193-1201 (1991);Mandriota et al., J. Biol. Chem., 270:9709-9716 (1995)). Thus uPA/uPARhas emerged as a new target for developing ananti-metastatic/anti-angiogenic therapy for cancer, where most studieshave been conducted (Fazioli et al., Trends Pharmacological Sci.,15:25-29 (1994).

However, the uPA/uPAR interaction goes far beyond localizing proteolysisat the cell surface. Independent of all proteolytic effects, the mereoccupation of uPAR by uPA induces, by indirect means, signaltransduction events leading to one or more of the following effects:mitogenesis (Rabbani et al., J Biol. Chem., 267:14151-56 (1992));expression of the c-fos gene (Dumler et al., FEBS Lett. 322:37-40(1994)); cysteine- and metalloprotease expression by macrophages (Rao etal., J. Clin. Invest. 96:465-74 (1995)): transfer of mechanical forceleading to increased cytoskeletal stiffness (Wang et al., Am. J.Physiol. 268:C1062-C1066 (1995)); endothelial cell migration (Odekon etal., J. Cellul. Physiol., 150:258-63 (1992)); endothelial cellmorphogenesis into tubular structures (Schnaper et al., J. Cellul.Physiol. 165:107-118 (1995)); and endothelial cell deformability andmotility (Lu et al., FEBS Lett. 380:21-24 (1996)). All of thesephenomena are blocked by blocking the access of uPA to uPAR. Anantagonist of uPAR that prevented the binding of uPA would thusinterfere with proteolytic activity by preempting uPA activation and,further, would greatly diminish uPAR's capacity for signal transduction.

The anti-angiogenic effects accompanying uPAR antagonism (Min et al.,Cancer Res., 56:2428-33 (1996)) should allow a uPAR antagonist to play arole in other diseases characterized by inappropriate angiogenesis, e.g.ocular angiogenesis leading to blindness. Furthermore, it is likely thata uPAR antagonist would also play a therapeutic role in inflammatorydiseases, for example, rheumatoid arthritis. (Ronday et al., Br. J.Rheum., 35:416-423 (1996).

One approach to drug therapy is to target uPA itself at its catalyticserine protease domain. Yang et al., Fibrinolysis, 6 (Suppl. 1):31-34,(1992). Amiloride (Vassalli et al., FEBS Lett., 214:187-191 (1987); andKellen et al., Anticancer Res. 8:1373-76 (1988)) and p-aminobenzamidine(Geratz et al., Thrombosis Res. 24:73-83 (1981); and Billstrom et al.,Int. J. Cancer, 61:542-47 (1995)) are competitive inhibitors of thissite and have anti-metastatic activity in vivo. Selective inhibition ofuPA as compared with other serine proteases, was evident inphenylguanidines (Yang et al., J. Med. Chem., 33:2956-61 (1990)) and,even more so, in benzo b!thiophene-2-carboxamidines (Bridges, Bioorganic& Medicinal Chemistry, 1:403-410 (1993); Towle et al., Cancer Res.,53:2553-59 (1993); and Rabbani et al., Int. J. Cancer, 63:840-45(1995)).

Towards defining the binding epitope for the uPA-uPAR interaction, itwas first shown that the amino terminal fragment of uPA (residues 1-135)that lacked the serine protease domain, sufficed for high affinity,sub-nanomolar binding. (Stoppelli et al., Proc. Natl. Acad. Sci. USA82:4939-43 (1985). Further work showed that the growth factor domainalone (residues 1-48) conferred this binding. (Robbiati et al.,Fibrinolysis, 4:53-60 (1990); and Stratton-Thomas et al., ProteinEngineering 8:463-70 (1995.)) Dano/ et al., in WO 90/12091 published 18Oct., 1990, discloses that the binding of uPA to uPAR could be preventedby administering a substance comprising a sequence identical orsubstantially identical to a uPAR binding site of uPA amino residues12-32. WO 94/28145, by Rosenberg and Stratton-Thomas, Dec. 8, 1994,discloses the preparation and use of de-fucosylated HuPA₁₋₄₈ thatprevents uPA binding to uPAR.

Earlier studies with peptide fragments within the growth factor domainhad showed that residues 20-30 conferred the specificity of binding, butthat residues 13-19 were needed in addition for residues 20-30 to attainthe proper binding conformation. Specifically, the peptide Ala¹⁹!uPA-(12-32) (SEQ ID NO:2, which contains two cysteines (the thirdcysteine being replaced by Ala to avoid undesired disulfide bondformations), in its open chain form prevented uPA binding to uPAR withan IC₅₀ of 100 nM. In its oxidized cyclic form, having an intrachaindisulfide bond between Cys¹³ and Cys³¹, the peptide prevented bindingwith an IC₅₀ of 40 nM. It was proposed that residues 13-19 might actindirectly to provide a scaffold that would help residues 20-30 attainthe correct binding conformation. Appella et al., J. Biol. Chem.,262:4437-40 (1987).

These results were partially confirmed when it was reported that, whilethe linear peptide 20-30 (SEQ ID NO:3) inhibited the binding of uPA touPAR with an IC₅₀ of 1,000 nM, the longer peptide 17-34 (SEQ ID NO:4)was significantly more potent, having an IC₅₀ of 100 nM. It was alsoshown that the corresponding longer peptide (17-34) derived from themouse sequence inhibited spontaneous metastasis of a murine Lewis Lungcarcinoma in mice, whereas the corresponding linear shorter peptide(20-30) (SEQ ID NO:3) had no effect. Kobayashi et al., Int. J. Cancer,57:727-33 (1994). WO 94/28014 by Rosenberg and Doyle, Dec. 8, 1994discloses the preparation and use of 25 random peptides displayed onbacteriophage which competed with the N-terminal fragment of uPA forbinding to uPAR with IC₅₀ values of 15 nM to >50 μM.

Most recently, Magdolen et al., "Systematic Mutational Analysis of theReceptor-binding Region of the Human Urokinase-type PlasminogenActivator", Eur. J. Biochem., 237:743-51 (1996), describesalanine-scanning mutagenesis of the binding loop of the amino-terminalfragment of uPA with the finding that Asn22, Lys23, Tyr24, Phe25, Ile28and Trp30 are important side chains that should be kept. Further,Magdolen et al., citing Hansen et al., Biochemistry, 33:4847-64 (1994),disclose that the region between Thr18 and Asn32 consists of a flexible,seven-residue omega loop that is forced into a ring-like structure.Although Cys19 and Cys31 are in close proximity to each other (0.61 nm),they do not form a disulfide bond with each other. Instead Cys19 forms adisulfide bond with Cys11, and Cys31 forms a bond with Cys13. See FIG. 2(SEQ ID NO:5). Accordingly, the uPAR binding site on uPA does not form asimple, small ring structure.

Some scientists have explored the possibility of cyclizing the one ormore of the growth factor domains of peptide analogues to increase theircompetitive binding activity, but not with any great success without atleast adding some other constraining-type modifications of thestructure. For example, in Chamberlin et al., J. Biol. Chem.,270:21062-21067 (1995), peptides constrained by the introduction of anintramolecular disulfide bond also required the substitution of anotherentity for proline in the peptide loop to achieve any significantactivity. Lougheed et al., Protein Sci., 4:773-80 (1995) found thatpeptides from the fifth EGF-like domain of thrombomodulin had very weakbiological activity that increased marginally (two-fold) by cyclization.The additional presence of a "tail" of amino acids and the deletion ofone of the amino acids were both found necessary and, even then, thebest peptide was only weakly active (text of micromolar range). Thus,cyclization per se conferred no significant activity. Further, othersworking in the thrombomodulin field have found that the number ofcrossing disulfide bonds in the fifth EGF-like domain is inversely,rather than directly, related to inhibitory potency. Hunter et al.,Protein Sci., 4:2129-37 (1995).

It has now been found by the present inventors that novel cyclicstructures derived from the peptide fragment 20-30, in which residue 20is covalently bonded to residue 30, do exhibit the ability to bind touPAR and are also antagonists of the binding of uPA to uPAR. Thesepeptides are shorter than either Ala¹⁹ !uPA-(12-32) (Appella et al.,supra.) or uPA17-34 (Kobayashi et al., supra.), but bind almost aseffectively. In contradiction of what was hitherto thought, it has beendiscovered that the eight amino acids N-terminal to Val20 in Ala¹⁹!uPA-(12-32) and the four amino acids CO-terminal to Trp30 in uPA17-34are not necessary for high binding affinity. While not wishing to bebound by any particular theory, it now appears that the minimal bindingepitope in urokinase-type plasminogen activator, which is needed forbinding to its receptor, is a loop of only eleven amino acids.

DISCLOSURE OF THE INVENTION

The invention is a cyclic peptide compound of Formula 1 or Formula 2##STR1## wherein, in Formula 1, all of X¹ through X¹¹ represent L-seriesamino acids and, in Formula 2, all of X¹ through X¹¹ represent D-seriesamino acids;

X¹ is Val, Pro, or Ala;

X² is Ser or Ala;

X³ is Asn or Gln;

X⁴ is Lys or His;

X⁵ is Tyr, Trp, Phe, substituted Phe, di-substituted Phe,homophenylalanine, β-(3-pyridyl)alanine, β-(2-thienyl)alanine,β-(1-naphthyl)alanine, or β-(2-naphthyl)alanine;

X⁶ is Tyr, Trp, Phe, substituted Phe, di-substituted Phe,homophenylalanine, β-(3-pyridyl)alanine, β-(2-thienyl)alanine,β-(1-naphthyl)alanine, or β-(2-naphthyl)alanine;

X⁷ is Ser or Ala;

X⁸ is Asn or Ala;

X⁹ is Ile, Leu, or Val;

X¹⁰ is His or Ala;

X¹¹ is Tyr, Trp, Phe, substituted Phe, di-substituted Phe,homophenylalanine, β-(3-pyridyl)alanine, β-(2-thienyl)alanine,β-(1-naphthyl)alanine, or β-(2-naphthyl)alanine; and

L is a linking unit, such that when X¹ and X¹¹ are linked, the lineardimension between the C.sup.α carbon of amino acid X¹ and the C.sup.αcarbon of amino acid X¹¹ is between about 4 and 12 Ångstrom units;

with the proviso that, when said compound is of Formula 1, L does notcomprise two cysteine units linked by a disulfide bond.

In other embodiments, the compounds of the invention are used in methodsand therapeutic compositions to inhibit the binding of uPA to uPAR,particularly in the treatment of cancer.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic representation of the pro-uPA molecule and itsmajor cleavage sites.

FIG. 2 (SEQ ID NO:5) shows the NH-terminal growth factor domain of humanuPA.

DETAILED DESCRIPTION OF THE INVENTION

The cyclic peptide compounds of the invention can be of either Formula 1or the corresponding retro-inverso cyclic peptide compounds of Formula2. When the cyclic peptide compounds of the invention are of Formula 1,all of X¹ through X¹¹ represent L-series natural amino acids. On theother hand, when the compounds of the invention are of Formula 2, all ofX¹ through X¹¹ represent D-series non-natural amino acids. Whether ofFormula 1 or Formula 2, however, when any one of X⁵, X⁶ or X¹¹ is asubstituted or disubstituted phenylalanine, the substituent is halo,such as 4-fluoro, 4-chloro, 4-bromo, or 3,4-dichloro; lower alkyl, suchas methyl, ethyl, isopropyl, tertiary butyl or n-pentyl; nitro; or thelike.

The linker moiety L in the compounds of the invention forms a bridgebetween X¹¹ and X¹, thus cyclizing the compound. The linker L can bealmost any divalent group which would set the linear dimension betweenthe C.sup.α carbon of amino acid X¹ and the C.sup.α carbon of amino acidX¹¹ between about 4 and 12 Ångstrom units, preferably between about 5and 10 Ångstrom units and, even more preferably, between about 6 and 8Ångstrom units.

COMPOUNDS OF FORMULA 1

In Formula 1, the amide bond, CO--NH, which links X¹ to X², is such thatthe C═O moiety is from the amino acid X¹ and the NH moiety is from theamino acid X². The same arrangement applies to the link between X² andX³, and so on. In other words, the peptide has X¹ as its N-terminus andX¹¹ as its C-terminus.

It should be noted, however, that L cannot comprise two cysteine unitslinked by a disulfide bond, be it unsubstituted, substituted at itsN-terminus with a group R¹, substituted at its C-terminus with a groupR², or modified at both N- and C-termini, respectively, with R¹ and R²,where R¹ is acetyl and R² is amino such that the C-terminus is a primarycarboxamide. Specific examples of such excluded L groups include:

--CO--CH(NH₂)--CH₂ --S--S--CH₂ --CH(COOH)--NH--;

--CO--CH(NHR¹)--CH₂ --S--S--CH₂ --CH(COOH)--NH--;

--CO--CH(NH₂)--CH₂ --S--S--CH₂ --CH(COR²)--NH--; and

--CO--CH(NHR¹)--CH₂ --S--S--CH₂ --CH(COR²)--NH--.

To prepare the compounds of Formula 1, L is chosen so as to provide, atone of its termini, a functional group that can be chemically bonded tothe carbon of the α-carboxyl group of the amino acid X¹¹ and, at itsother terminus, a functional group that can be chemically bonded to theα-amino nitrogen atom of the amino acid X¹.

Alternatively, the linear peptide X¹ -X² -X³ -X⁴ -X⁵ -X⁶ -X⁷ -X⁸ -X⁹-X¹⁰ -X¹¹ (SEQ ID NO:7) can be synthesized with an extension at its X¹¹terminus comprising a portion of the final linker group, i.e., L_(b),and later, after synthesis of the desired peptide chain, the X¹ terminuscan be extended with a group L_(a), to give the compound L_(a) -X¹ -X²-X³ -X⁴ -X⁵ -X⁶ -X⁷ -X⁸ -X⁹ -X¹⁰ -X¹¹ -L_(b). The free ends of L_(a) andL_(b) can then be chemically bonded to each other. In this way, thelinker L can be formed during the cyclization step from pre-attachedfragments L_(a) and L_(b). In the examples given below for L, thedirection of L, reading left to right, is from to X¹ to X¹¹, i.e., theCO-terminus of L is connected to X¹, and the NH-terminus of L isconnected to X¹¹.

Typical examples of useful L groups for Formula 1 include the following:

--CO--CH₂ --CH₂ --CH₂ --S--CH₂ --CH(COOH)--NH--;

--CO--CH₂ --CH₂ --CH₂ --CH₂ --S--CH₂ --CH(COOH)--NH--;

--CO--CH(NH₂)--CH₂ --S--CH₂ --CH₂ --CH₂ --NH--;

--CO--CH₂ --CH₂ --S--CH₂ --CH₂ --CH₂ --NH--;

--CO--CH₂ --CH₂ --CH₂ --CH₂ --CH₂ --CH₂ --NH--;

--CO--CH₂ --CH₂ --S--CH₂ --CH₂ --CH(COOH)--NH--;

--CO--CH₂ --CH₂ --CH₂ --CH₂ --CH₂ --CH(COOH)--NH--;

--CO--CH₂ --S--CH₂ --CH(COOH)--NH--;

--CO--CH₂ --S--CH₂ --CH₂ --CH(COOH)--NH--; and

--CO--CH₂ -meta-phenylene-CH₂ --NH--.

When L contains a cysteine or a homocysteine residue, the configurationof the enantiomeric center of such a residue can be either L- or D-.

COMPOUNDS OF FORMULA 2 (RETRO-INVERSO COMPOUNDS)

When the cyclic peptide compound of the invention is of Formula 2, theamino acids X¹ -X¹¹ are non-natural, "D-series" peptides having anenantiomeric configuration opposite to that the of L-series naturalamino acids described above for Formula 1. In Formula 2, the amide bondCO--NH, which links X¹¹ to X¹⁰, is such that the C═O moiety is from theamino acid X¹¹ and the NH moiety is from the amino acid X¹⁰. The samearrangement applies to the link between X¹⁰ and X⁹ and so on. In otherwords, the peptide has X¹¹ as its N-terminus and X¹ as its C-terminus.

In the above structure of Formula 2, the moiety L is chosen so as toprovide, at one of its termini, a functional group that can bechemically bonded to the α-amino nitrogen atom of the X¹¹ residue,rather than the α-carboxyl group and, at its other terminus, afunctional group that can be chemically bonded to the carbon of theα-carboxyl group of the of the amino acid X¹, rather than the α-aminonitrogen atom, as described above.

Also as described above, the linear peptide X¹ -X² -X³ -X⁴ -X⁵ -X⁶ -X⁷-X⁸ -X⁹ -X¹⁰ -X¹¹ (SEQ ID NO:7) can be synthesized at its X¹ terminuswith a group L_(b) and, at its X¹¹ terminus, with a group L_(a) to givethe compound L_(b) -X¹ -X² -X³ -X⁴ -X⁵ -X⁶ -X⁷ -X⁸ -X⁹ -X¹⁰ -X¹¹ -L_(a),such that the free ends of L_(a) and L_(b) can be chemically bonded toform the cyclic retro-inverso compounds of the invention.

In the examples given below for L, the direction of L, reading left toright, is from to X¹ to X¹¹, that is, the NH-terminus of L is covalentlybonded to X¹, and the CO terminus of L is connected to X¹¹. When theselinkers contain a cysteine or a homocysteine residue, the configurationof the enantiomeric center of such a residue can be either the D- or L-form.

Examples of useful L groups for the retro-inverso peptides of Formula 2include:

--NH--CH(COOH)--CH₂ --S--CH₂ --CH₂ --CH₂ --CO--;

--NH--CH(COOH)--CH₂ --S--CH₂ --CH₂ --CH₂ --CH₂ --CO--;

--NH--CH₂ --CH₂ --CH₂ --S--CH₂ --CH(NH₂)--CO--;

--NH--CH₂ --CH₂ --CH₂ --S--CH₂ --CH₂ --CO--;

--NH--CH₂ --CH₂ --CH₂ --CH₂ --CH₂ --CH₂ --CO--;

--NH--CH(COOH)--CH₂ --CH₂ --S--CH₂ --CH₂ --CO--;

--NH--CH(COOH)--CH₂ --CH₂ --CH₂ --CH₂ --CH₂ --CO--;

--NH--CH(COOH)--CH₂ --S--CH₂ --CO--;

--NH--CH(COOH)--CH₂ --CH₂ --S--CH₂ --CO--; and

--NH--CH₂ -meta-phenylene-CH₂ --CO--.

GENERAL CHEMICAL SYNTHETIC PROCEDURES

When the peptides of the invention are not prepared using recombinantDNA technology, they are preferably prepared using solid-phasesynthesis, such as that generally described by Merrifield, J. Amer.Chem. Soc., 85:2149-54 (1963), although other equivalent chemicalsyntheses known in the art are also useful. Solid-phase peptidesynthesis may be initiated from the C-terminus of the peptide bycoupling a protected α-amino acid to a suitable resin. Such a startingmaterial can be prepared by attaching an α-amino-protected amino acid byan ester linkage to a chloromethylated resin or to a hydroxymethylresin, or by an amide bond to a BHA resin or MBHA resin.

The preparation of the hydroxymethyl resin is described by Bodansky etal., Chem. Ind., 38:1597-98 (1966). Chloromethylated resins arecommercially available from BioRad Laboratories, Richmond, Calif. andfrom Lab. Systems, Inc. The preparation of such a resin is described byStewart et al., "Solid Phase Peptide Synthesis" (Freeman & Co., SanFrancisco 1969), Chapter 1, 1-6. BHA and MBHA resin supports arecommercially available and are generally used only when the desiredpolypeptide being synthesized has an unsubstituted amide at theC-terminus.

The amino acids X¹ through X¹¹ can be coupled to the growing peptidechain using techniques well known in the art for the formation ofpeptide bonds. For example, one method involves converting the aminoacid to a derivative that will render the carboxyl group of the aminoacid more susceptible to reaction with the free N-terminal amino groupof the growing peptide chain. Specifically, the C-terminal of theprotected amino acid can be converted to a mixed anhydride by thereaction of the C-terminal with ethyl chloroformate, phenylchloroformate, sec-butyl chloroformate, isobutyl chloroformate, orpivaloyl chloride or the like acid chlorides. Alternatively, theC-terminal of the amino acid can be converted to an active ester, suchas a 2,4,5-trichlorophenyl ester, a pentachlorophenyl ester, apentafluorophenyl ester, a p-nitrophenyl ester, a N-hydroxysuccinimideester, or an ester formed from 1-hydroxybenzotriazole.

Another coupling method involves the use of a suitable coupling agent,such as N,N'dicyclohexylcarbodiimide or N,N'-diisopropylcarbodiimide.Other appropriate coupling agents, apparent to those skilled in the art,are disclosed in Gross et al., The Peptides: Analysis, Structure,Biology, Vol. I:, "Major Methods of Peptide Bond Formation" (AcademicPress 1979), the disclosure of which is hereby incorporated byreference.

It will be recognized that the a-amino group of each amino acid employedin the peptide synthesis must be protected during the coupling reactionto prevent side reactions involving their active α-amino function. Itshould also be recognized that certain amino acids contain reactiveside-chain functional groups (e.g. sulflydryl, amino, carboxyl, andhydroxyl) and that such functional groups must also be protected withsuitable protecting groups to prevent a chemical reaction from occurringat either (1) the α-amino group site or (2) a reactive side chain siteduring both the initial and subsequent coupling steps.

In the selection of a particular protecting group to be used insynthesizing the peptides, the following general rules are typicallyfollowed. Specifically, an α-amino protecting group (a) should renderthe a-amino function inert under the conditions employed in the couplingreaction, (b) should be readily removable after the coupling reactionunder conditions that will not remove side-chain protecting groups andwill not alter the structure of the peptide fragment, and (c) shouldsubstantially reduce the possibility of racemization upon activation,immediately prior to coupling.

On the other hand, a side-chain protecting group (a) should render theside chain functional group inert under the conditions employed in thecoupling reaction, (b) should be stable under the conditions employed inremoving the α-amino protecting group, and (c) should be readilyremovable from the desired fully-assembled peptide under reactionconditions that will not alter the structure of the peptide chain.

It will be apparent to those skilled in the art that the protectinggroups known to be useful for peptide synthesis will vary in reactivitywith the agents employed for their removal. For example, certainprotecting groups, such as triphenylmethyl and2-(p-biphenyl)isopropyloxycarbonyl, are very labile and can be cleavedunder mild acid conditions. Other protecting groups, such ast-butyloxycarbonyl (BOC), t-amyloxycarbonyl, adamantyl-oxycarbonyl, andp-methoxybenzyloxycarbonyl, are less labile and require moderatelystrong acids for their removal, such as trifluoroacetic, hydrochloric,or boron trifluoride in acetic acid. Still other protecting groups, suchas benzyloxycarbonyl (CBZ or Z), halobenzyloxycarbonyl,p-nitrobenzyloxycarbonyl cycloalkyloxycarbonyl, andisopropyloxycarbonyl, are even less labile and require even strongeracids, such as hydrogen fluoride, hydrogen bromide, or borontrifluoroacetate in trifluoroacetic acid, for their removal. Suitableprotecting groups, known in the art are described in Gross et al., ThePeptides: Analysis, Structure, Biology,. Vol 3: "Protection ofFunctional Groups in Peptide Synthesis" (Academic Press 1981).

Among the classes of amino acid protecting groups useful for protectingthe α-amino group or for protecting a side chain group are included thefollowing.

(1) For an α-amino group, three typical classes of protecting groupsare: (a) aromatic urethane-type protecting groups, such asfluorenylmethyloxycarbonyl (FMOC), CBZ, and substituted CBZ, such as,e.g., p-chlorobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl,p-bromobenzyloxycarbonyl, and p-methoxybenzyloxycarbonyl,o-chlorobenzyloxycarbonyl, 2,4-dichlorobenzyloxycarbonyl,2,6-dichlorobenzyloxycarbonyl, and the like; (b) aliphatic urethane-typeprotecting groups, such as BOC, t-amyloxycarbonyl, isopropyloxycarbonyl,2-(p-biphenyl)-isopropyloxycarbonyl, allyloxycarbonyl and the like; and(c) cycloalkyl urethane-type protecting groups, such ascyclopentyloxycarbonyl, adamantyloxycarbonyl, and cyclohexyloxycarbonyl.The preferred α-amino protecting groups are BOC and FMOC.

(2) For the side chain amino group present in Lys, protection may be byany of the groups mentioned above in (1) such as BOC,2-chlorobenzyloxycarbonyl and the like.

(3) For the guanidino group of Arg, protection may be provided by nitro,tosyl, CBZ, adamantyloxycarbonyl,2,2,5,7,8-pentamethylchroman-6-sulfonyl,2,3,6-trimethyl-4-methoxyphenylsulfonyl, or BOC groups.

(4) For the hydroxyl group of Ser, Thr, or Tyr, protection may be, forexample, by t-butyl; benzyl (BZL); or substituted BZL, such asp-methoxybenzyl, p-nitrobenzyl, p-chlorobenzyl, o-chlorobenzyl, and2,6-dichlorobenzyl.

(5) For the carboxyl group of Asp or Glu, protection may be, forexample, by esterification using such groups as BZL, t-butyl,cyclohexyl, cyclopentyl, and the like.

(6) For the imidazole nitrogen of His, the benzyloxymethyl (BOM) ortosyl moiety is suitably employed as a protecting group.

(7) For the phenolic hydroxyl group of Tyr, a protecting group such astetrahydropyranyl, tert-butyl, trityl, BZL, chlorobenzyl, 4-bromobenzyl,and 2,6-dichlorobenzyl are suitably employed. The preferred protectinggroup is bromobenzyloxycarbonyl.

(8) For the side chain amino group of Asn or Gln, xanthyl (Xan) ispreferably employed.

(9) For Met, the amino acid is preferably left unprotected.

(10) For the thio group of Cys, p-methoxybenzyl is typically employed.

The first C-terminal amino acid of the growing peptide chain, e.g., Lys,is typically protected at the N-amino position by an appropriatelyselected protecting group such as BOC. TheBOC-Lys-(2-chlorobenzyloxycarbonyl)-OH group can be first coupled to abenzylhydrylamine resin using isopropylcarbodiimide at about 25° C. fortwo hours with stirring or to a chloromethylated resin according to theprocedure set forth in Horiki et al., Chemistry Letters, 165-168 (1978).Following the coupling of the BOC-protected amino acid to the resinsupport, the α-amino protecting group is usually removed, typically byusing trifluoroacetic acid (TFA) in methylene chloride or TFA alone. Theα-amino group de-protection reaction can occur over a wide range oftemperatures, but is usually carried out at a temperature between about0° C. and room temperature.

Other standard α-amino group de-protecting reagents, such as HCl indioxane, and conditions for the removal of specific α-amino protectinggroups are within the skill of those working in the art, such as thosedescribed in Lubke et al., Chemie und Biochemie der Aminosauren, Peptideund Proteine I, Chapter II-1, 102-117 (Georg Thieme Verlag Stuttgart1975), which is hereby incorporated by reference. Following the removalof the α-amino protecting group, the unprotected α-amino group,generally still side-chain protected, can be coupled in a stepwisemanner in the intended sequence.

An alternative to the stepwise approach is the fragment condensationmethod in which pre-formed peptides of short length, each representingpart of the desired sequence, are coupled to a growing chain of aminoacids bound to a solid phase support. For the stepwise approach, aparticularly suitable coupling reagent is N,N'-dicyclohexylcarbodiimideor diisopropylcarbodiimide. Also, for the fragment approach, theselection of the coupling reagent, as well as the choice of thefragmentation pattern needed to couple fragments of the desired natureand size are important for success and are known to those skilled in theart.

Each protected amino acid or amino acid sequence is usually introducedinto the solid-phase reactor in amounts in excess of stoichiometricquantities, and the coupling is suitably carried out in an organicsolvent, such as dimethylformamide (DMF), CH₂ Cl₂ or mixtures thereof.If incomplete coupling occurs, the coupling procedure is customarilyrepeated before removal of the N-amino protecting group in preparationfor coupling to the next amino acid. Following the removal of theα-amino protecting group, the remaining α-amino and side-chain-protectedamino acids can be coupled in a stepwise manner in the intendedsequence. The success of the coupling reaction at each stage of thesynthesis may be monitored. A preferred method of monitoring thesynthesis is by the ninhydrin reaction, as described by Kaiser et al.,Anal. Biochem., 34:595 (1970). The coupling reactions can also beperformed automatically using well-known commercial methods and devices,for example, a Beckman 990 Peptide Synthesizer.

Upon completion of the desired peptide sequence, the protected peptidemust be cleaved from the resin support, and all protecting groups mustbe removed. The cleavage reaction and removal of the protecting groupsis suitably accomplished concomitantly or consecutively withde-protection reactions. When the bond anchoring the peptide to theresin is an ester bond, it can be cleaved by any reagent that is capableof breaking an ester linkage and of penetrating the resin matrix. Oneespecially useful method is by treatment with liquid anhydrous hydrogenfluoride. This reagent will usually not only cleave the peptide from theresin, but will also remove all acid-labile protecting groups and, thus,will directly provide the fully de-protected peptide. When additionalprotecting groups that are not acid-labile are present, additionalde-protection steps must be carried out. These steps can be performedeither before or after the hydrogen fluoride treatment described above,according to specific needs and circumstances.

When a chloromethylated resin is used, the hydrogen fluoridecleavage/deprotection treatment generally results in the formation ofthe free peptide acids. When a benzhydrylamine resin is used, thehydrogen fluoride treatment generally results in the free peptideamides. Reaction with hydrogen fluoride in the presence of anisole anddimethylsulfide at 0° C. for one hour will typically remove theside-chain protecting groups and, concomitantly, release the peptidefrom the resin.

When it is desired to cleave the peptide without removing protectinggroups, the protected peptide-resin can be subjected to methanolysis,thus yielding a protected peptide in which the C-terminal carboxyl groupis methylated. This methyl ester can be subsequently hydrolyzed undermild alkaline conditions to give the free C-terminal carboxyl group. Theprotecting groups on the peptide chain can then be removed by treatmentwith a strong acid, such as liquid hydrogen fluoride. A particularlyuseful technique for methanolysis is that of Moore et al., Peptides,Proc. Fifth Amer. Pept. Symp., 518-521 (Goodman et al., eds., 1977), inwhich the protected peptide-resin is treated with methanol and potassiumcyanide in the presence of a crown ether.

Other methods for cleaving a protected peptide from the resin when achloromethylated resin is employed include (1) ammonolysis and (2)hydrazinolysis. If desired, the resulting C-terminal amide or hydrazidecan be hydrolyzed to the free C-terminal carboxyl moiety, and theprotecting groups can be removed conventionally. The protecting grouppresent on the N-terminal α-amino group may be removed either before, orafter, the protected peptide is cleaved from the support.

Purification of the polypeptides of the invention is typically achievedusing chromatographic techniques, such as preparative HPLC (includingreverse phase HPLC), gel permeation, ion exchange, partitionchromatography, affinity chromatography (including monoclonal antibodycolumns), and the like, or other conventional techniques such ascountercurrent distribution or the like.

The compounds of the invention can be easily tested for their ability toinhibit the binding of pro-uPA to uPAR in a competitive ligand-bindingassay. The assay is preferably a solid phase immunoassay that uses therecombinant soluble human urokinase receptor (uPAR) derived from Chinesehamster ovary cells coated onto the walls of test wells to act ascapture molecules. The compound being tested competes with pro-uPA forthe binding site on uPAR. The amount of pro-uPA that binds to the uPARon the walls of the test wells can be detected with a biotinylatedmonoclonal anti-uPA antibody that is, in turn, recognized bystreptavidin-horse radish peroxidase. The addition of perborate and3,3',5,5'-tetramethylbenzidine ("TMB") as a substrate allows theperoxidase to generate a blue-colored product, thus providing acolorimetric signal. The sensitivity of the assay is preferablyincreased even further by adding sulfuric acid to the test solution,which provides a yellow color that can be easily read out.

In general, the greater the binding of the test compound to uPAR, thegreater the exclusion of pro-uPA from binding, and the smaller will bethe generated optical signal. The specificity of this capture-type assayis inherent in the purity of the uPAR being used, which itself may betested by SDS-PAGE, non-reducing Western blot analysis (clean, singleband at 46 kD, the known molecular weight of intact uPAR).

The detection antibody in the assay recognizes an epitope within thekringle domain of uPA and, thus, is suitable for measuring thecompetitive binding of polypeptides that lack the kringle domain, suchas the growth factor domain of uPA or peptide derivatives representing apart of the growth factor domain, for example, the compounds of theinvention. This assay has been validated against three different formsof uPA: (1) pro-uPA, also known as single-chain uPA, (2) highmolecular-weight uPA, also known as two-chain uPA, and (3) lowmolecular-weight uPA.

A typical assay procedure is as follows:

uPAR is coated onto microtest wells in 96-well plates. 0.8 nM (160ng/mL) pro-uPA is incubated with serially diluted concentrations of thetest compound. Both pro-uPA and the compound being tested are diluted inphosphate-buffered saline (PBS) with 0.1% Triton X-100 and 1.0% bovineserum albumin (pH 7.4). The total volume per well of pro-uPA/testcompound mixture is 50 μL, and ligands are allowed to bind approximately16 hours overnight at 4° C.

The wells are washed four times with saline wash buffer (100 mMtrisodium phosphate, 150 mM sodium chloride, pH 7.4, containing 1%Triton X-100, and 0.025% sodium azide). The detection antibody solution(50 μL) is added, and the mixture is kept at room temperature for onehour. The wells are washed with wash buffer.

Streptavidin-horse radish peroxidase (50 μL) is next added and incubatedfor one hour at room temperature. The wells are again washed with washbuffer. Finally, the peroxidase substrate TMB (50 μL) and perborate areadded and allowed to react for 20 minutes to generate a blue color.Sulfuric acid (0.5 M, 50 μL) is added to yield a yellow color. Theabsorbance of the yellow color is then read out at 450 nanometers on anMRX microplate reader made by Dynatech Laboratories. Each test compoundis assayed in triplicate at five different concentrations.

The inhibition of binding of pro-uPA to uPAR is usually dose-related,such that the concentration of the test compound necessary to produce a50% inhibition of binding (the IC₅₀ value) is easily determined. Ingeneral, the compounds of the invention have IC₅₀ values of less thanabout 10⁻⁵ molar, i.e., less than about 10 μM. Preferably, the compoundsof the invention have IC₅₀ values of less than about 10⁻⁶ molar, i.e.,less than about 1 μM and, even more preferably, less than about 10⁻⁷molar.

ADMINISTRATION AND USE

The cyclic peptide compounds of the invention that may be employed inthe pharmaceutical compositions of the invention include all of thosecompounds described above, as well as the pharmaceutically acceptablesalts of these compounds. Pharmaceutically acceptable acid additionsalts of the compounds of the invention containing a basic group areformed where appropriate with strong or moderately strong, non-toxic,organic or inorganic acids in the presence of a basic amine by methodsknown to the art. Exemplary of the acid addition salts that are includedin this invention are maleate, fumarate, lactate, oxalate,methanesulfonate, ethanesulfonate, benzenesulfonate, tartrate, citrate,hydrochloride, hydrobromide, sulfate, phosphate and nitrate salts.

Pharmaceutically acceptable base addition salts of compounds of theinvention containing an acidic group are prepared by known methods fromorganic and inorganic bases and include, for example, nontoxic alkalimetal and alkaline earth bases, such as calcium, sodium, potassium andammonium hydroxide; and nontoxic organic bases such as triethylamine,butylamine, piperazine, and tri(hydroxymethyl)methylamine.

As stated above, the compounds of the invention possess the ability toinhibit uPA formation, a property that may express itself in the form ofanti-tumor activity. A compound of the invention may be active per se,or it may be a pro-drug that is converted in vivo to an active compound.

The compounds of the invention, as well as the pharmaceuticallyacceptable salts thereof, may be incorporated into convenient dosageforms, such as capsules, tablets or injectable preparations. Solid orliquid pharmaceutically acceptable carriers may be employed. Preferably,the compounds of the invention are administered systemically, e.g., byinjection. When used, injection may be intravenous, subcutaneous,intramuscular, or even intraperitoneal. Injectables can be prepared inconventional forms, either as solutions or suspensions, solid formssuitable for solution or suspension in liquid prior to injection, or asemulsions.

Solid carriers include starch, lactose, calcium sulfate dihydrate, terraalba, sucrose, talc, gelatin, agar, pectin, acacia, magnesium stearateand stearic acid. Liquid carriers include syrup, peanut oil, olive oil,saline, water, dextrose, glycerol and the like. Similarly, the carrieror diluent may include any prolonged release material, such as glycerylmonostearate or glyceryl distearate, alone or with a wax. When a liquidcarrier is used, the preparation may be in the form of a syrup, elixir,emulsion, soft gelatin capsule, sterile injectable liquid (e.g., asolution), such as an ampoule, or an aqueous or non-aqueous liquidsuspension. A summary of such pharmaceutical compositions may be found,for example, in Remington's Pharmaceutical Sciences, Mack PublishingCompany, Easton Pa. (Gennaro 18th ed. 1990).

The pharmaceutical preparations are made following conventionaltechniques of a pharmaceutical chemist involving such steps as mixing,granulating and compressing, when necessary for tablet forms, or mixing,filling and dissolving the ingredients, as appropriate, to give thedesired products for oral, parenteral, topical, transdermal,intravaginal, intranasal, intrabronchial, intraocular, intraaural andrectal administration. Of course, these compositions may also containminor amounts of nontoxic auxiliary substances such as wetting oremulsifying agents, pH buffering agents and so forth.

The compositions of the invention may further comprise one or more othercompounds that are anti-tumor agents, such as mitotic inhibitors, e.g.,vinblastine; alkylating agents, e.g., methotrexate, pritrexim ortrimetrexate); antimetabolites, e.g., 5-fluorouracil and cytosinearabinoside; intercalating antibiotics, e.g., adriamycin and bleomycin);enzymes, e.g., asparaginase; topoisomerase inhibitors, e.g., etoposide;or biological response modifiers, e.g., interferon.

The composition of the invention may also comprise one or more othercompounds, including antibacterial, anti-fungal, anti-parasitic,anti-viral, anti-psoriatic and anti-coccidial agents. Exemplaryantibacterial agents include, for example, sulfonamides such assulfamethoxazole, sulfadiazine, sulfameter or sulfadoxine; DHFRinhibitors such as trimethoprim, bromodiaprim or trimetrexate;penicillins; cephalosporins; aminoglycosides; bacteriostatic inhibitorsof protein synthesis; the quinolonecarboxylic acids and their fusedisothiazole analogs; and the like.

Another aspect of the invention relates to a therapeutic processpreventing the binding of uPA to uPAR, which process comprisesadministering to a vertebrate host, such as a mammal or bird, an amounteffective to inhibit the binding of uPA to uPAR according to the presentinvention. The compounds of the invention are particularly useful in thetreatment of mammalian hosts, such as human hosts and other animal hostslikely to be provided with a veterinarian's care. Further, the peptidesof the invention that are capable of carrying a suitable radioactivefluorogenic, chromogenic, or chemical label can also be used toquantitate uPAR levels in tissue samples and has use, therefore, indiseases where the receptor plays a pathological role.

Any of the cyclic peptide compounds described above, or pharmaceuticallyacceptable salts thereof, may be used in the therapeutic process of theinvention. The compounds may be administered in the therapeutic processof the invention in the form of a pharmaceutically acceptablecomposition comprising a diluent or carrier, such as those describedabove. Doses of the compounds preferably include pharmaceutical dosageunits comprising an efficacious quantity of active compound. By anefficacious quantity is meant a quantity sufficient to inhibit thebinding of uPA to uPAR and derive the beneficial effects therefromthrough administration of one or more of the pharmaceutical dosageunits.

The quantity of compound to be administered depends on the choice ofactive ingredient, the conditions to be treated, the mode ofadministration, the individual subject, and the judgment of thepractitioner. Depending on the specificity of the preparation, smalleror larger doses may be needed. An exemplary daily dosage unit for avertebrate host comprises an amount of up to about 5,000 mg of activecompound per square meter of the body area of the vertebrate host.Dosages in the range of about 0.05-10 mg/kg are suggested for systemicadministration. Dosages in the range of about 0.01-20% concentration ofactive ingredient, preferably 1-5%, are suggested for topicaladministration. A total daily dosage in the range of about 10-300 mg issuggested for oral administration. The foregoing ranges are, however,merely suggestive, as the number of variables in regard to an individualtreatment regime is large, and considerable excursions from theserecommended values are expected.

The selected dose may be administered to a warm-blooded animal ormammal, for example, a human patient, in need of treatment mediated byinhibition of the binding of uPA to uPAR by any known method ofadministration, including topically or transdermally, e.g., as anointment, cream or gel; orally; rectally, e.g., as a suppository;parenterally, by injection or continuously by infusion; intravaginally;intranasally; intrabronchially; intra-aurally; or intraocularly.

The cyclic peptide compounds of the invention may be furthercharacterized as producing any one or more of inhibitor effect on uPAbinding to uPAR, a proteolysis inhibitor effect, an inhibitory effect onprogrammed gene expression; an inhibitory effect on cell motility,migration and morphogenesis; an effect of slowing the activation ofpro-growth factors to the active form of the growth factor; ananti-angiogenesis effect; an inhibitory effect on tumor metastasis; alessening of retinal neovascularization; and a protective effect againstinflammatory diseases such as arthritis. The compounds are especiallyuseful in producing an anti-tumor effect in a vertebrate host harboringa tumor.

Further details of the production and purification of the compounds ofthe invention are given in the following illustrative specific example,which is not in any way intended to limit the scope of the invention.

EXAMPLE 1 Synthesis of (SEQ ID NO:8) ##STR2## The starting material wasBOC-L-Cys(S-p-Methoxybenzyl)-O-resin, substituted at a level of 0.84milli-equivalent per gram of resin. Each of the remaining L-amino acidswas added in sequence in a synthesis cycle consisting of:

1. TFA De-protection

The BOC protecting group was removed from the α-amino nitrogen of thestarting material by treating the resin with 50% trifluoroacetic acid(TFA) in dichloromethane (DCM) (two to three volumes per resin volume).The mixture was stirred at room temperature for 30 minutes and thendrained. The resin was then washed once with an equal volume ofisopropanol for one minute and washed twice with an equal volume ofmethanol, each wash taking one minute.

2. Coupling

The de-protected resin was washed twice with an equal volume of 10%triethylamine in DCM, each wash taking one minute, and washed twice withan equal volume of methanol, each wash taking one minute, and washedtwice with an equal volume of DCM, each wash taking one minute. ABOC-protected amino acid (three equivalents, dissolved in DCM or in amixture of DCM and N,N'-dimethylformamide (DMF)) and1-hydroxybenzotriazole (1M solution in DMF, three equivalents) was addedto the resin, and the mixture was stirred for a few seconds.Dicyclohexylcarbodiimide ("DCC") (1M solution in DCM, three equivalents)was then added, and the whole mixture was stirred for 60-120 minutes.The resin was washed twice with an equal volume of methanol and thenwashed twice with an equal volume of DCM. A small sample was taken for aninhydrin test to assess the completeness of coupling. Generally, ifincomplete, the coupling step 2 is repeated. If complete, the synthesisis continued with the capping step 3.

In the case of each of the five N-terminal amino acids,Val-Ser-Asn-Lys-Tyr (SEQ ID NO:9), coupling was only partially completeand, therefore, was repeated at least once. The coupling of4-bromobutyric acid was first attempted with DCC, as described above forthe coupling step 2, but was incomplete. Three manual repeat couplingswere performed using the symmetric anhydride method, thepentafluorophenylester method and, finally, with bromobutyryl chloride.A ninhydrin test following these procedures indicated virtually completecoupling.

All amino acids were used as α-BOC derivatives. Side chain protectinggroups were as follows:

    ______________________________________                                        Cysteine            p-Methoxybenzyl                                           Tryptophan          N-Formyl                                                  Histidine           Benzyloxymethyl                                           Asparagine          Xanthyl                                                   Serine              O-benzyl                                                  Tyrosine            2-Bromo-Z                                                 Lysine              2-Chloro-Z                                                ______________________________________                                    

Before coupling with bromobutyric acid, the formyl group of thetryptophan residue was removed by treatment with 20% piperidine in DMFat room temperature for 30 minutes.

3. Capping

The resin was stirred with an equal volume of acetic anhydride (20%solution in DCM) for 5 minutes at room temperature. The resin was washedtwice with an equal volume of methanol and then washed twice with anequal volume of DCM.

4. HF Cleavage

The resin bearing the desired amino acid sequence (1.0 gram) was placedin a Teflon reaction vessel, and anhydrous anisole (1 mL) was added. Thevessel was cooled with liquid N₂, and anhydrous HF (10 mL) was distilledinto it. The temperature was raised with ice water to 0° C. The mixturewas stirred at this temperature for one hour, and then the HF wasdistilled off at 0° C. The residue was washed with anhydrous ether, andthe peptide was extracted with a 1:1 mixture of CH₃ CN:H₂ O.

5. Cyclization

The above peptide solution was diluted with H₂ O to a final volume ofapproximately 1000 mL, and the pH was adjusted with NaHCO₃ to 8.0. Thecyclization was monitored by analytical HPLC. After 2-3 days, no furthershift was observed in the chromatogram, and the cyclization was judgedcomplete.

6. Purification

The turbid cyclization reaction mixture was filtered through a 1 μMfilter. The filtrate was adjusted to pH 4.0 and loaded onto a Waters C18preparative column (2 inches diameter, 15-20 μm particle size, 300 Åpore size). The loaded column was eluted with a two-component eluentapplied as a linear gradient, starting with 15% of solution A insolution B and finishing with 40% of solution A in solution B. SolutionA was 0.1% TFA in H₂ O, and solution B was 0.1% TFA in CH₃ CN. Fractionsexhibiting purity equal to or better than that desired were pooled andlyophilized to render the purified final product as the trifluoroacetatesalt.

    __________________________________________________________________________    #             SEQUENCE LISTING                                                - (1) GENERAL INFORMATION:                                                    -    (iii) NUMBER OF SEQUENCES: 9                                             - (2) INFORMATION FOR SEQ ID NO:1:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 44 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:                                 - Ser Asn Glu Leu His Gln Val Pro Ser Asn Cy - #s Asp Cys Leu Asn Gly         #                15                                                           - Gly Thr Cys Val Ser Asn Lys Tyr Phe Ser As - #n Ile His Trp Cys Asn         #            30                                                               - Cys Pro Lys Lys Phe Gly Gly Gln His Cys Gl - #u Ile                         #        40                                                                   - (2) INFORMATION FOR SEQ ID NO:2:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 21 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: both                                                  -     (ix) FEATURE:                                                                     (A) NAME/KEY: Disulfide-bo - #nd                                              (B) LOCATION: 2..20                                                 #/note= "present in oxidized cyclic                                                          form"                                                          -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:                                 - Asp Cys Leu Asn Gly Gly Thr Ala Val Ser As - #n Lys Tyr Phe Ser Asn         #                15                                                           - Ile His Trp Cys Asn                                                                     20                                                                - (2) INFORMATION FOR SEQ ID NO:3:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 11 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:                                 - Val Ser Asn Lys Tyr Phe Ser Asn Ile His Tr - #p                             #                10                                                           - (2) INFORMATION FOR SEQ ID NO:4:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 18 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:                                 - Gly Thr Cys Val Ser Asn Lys Tyr Phe Ser As - #n Ile His Trp Cys Asn         #                15                                                           - Cys Pro                                                                     - (2) INFORMATION FOR SEQ ID NO:5:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 49 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: Disulfide-bo - #nd                                              (B) LOCATION: group(11..19 - #, 13..31, 33..42)                     -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:                                 - Ser Asn Glu Leu His Gln Val Pro Ser Asn Cy - #s Asp Cys Leu Asn Gly         #                15                                                           - Gly Thr Cys Val Ser Asn Lys Tyr Phe Ser As - #n Ile His Trp Cys Asn         #            30                                                               - Cys Pro Lys Lys Phe Gly Gly Gln His Cys Gl - #u Ile Asp Lys Ser Lys         #        45                                                                   - Thr                                                                         - (2) INFORMATION FOR SEQ ID NO:6:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 11 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: circular                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/note= "Val, Pro or Ala"MATION:                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                     #7)       (B) LOCATION: group(2,                                              #/note= "Ser or Ala"INFORMATION:                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                     #/note= "Ans or Gln"INFORMATION:                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                     #/note= "Lys or His"INFORMATION:                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                     #6, 11)   (B) LOCATION: group(5,                                              #/note= "Tyr, Trp, Phe, substitued                                                           Phe, di-s - #ubstituted Phe, homophenylalanine,                               beta-(3-pyri - #dyl)alanine, beta-(2-thienyl)alanine,                         beta-(1-naph - #thyl)alanine or beta-(2-naphthyl)alanine"      -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 8                                                     #/note= "Asn or Ala"INFORMATION:                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 9                                                     #/note= "Ile, Leu or Val"MATION:                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                    #/note= "His or Ala"INFORMATION:                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: Cross-links                                                     (B) LOCATION: join(1, 1 - #1)                                       #/label= L(D) OTHER INFORMATION:                                              #12inear dimension of linkage is between 4 and                                #units"        Angstrom                                                       -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:                                 - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a                             #                10                                                           - (2) INFORMATION FOR SEQ ID NO:7:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 11 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 1                                                     #/note= "Val, Pro or Ala"MATION:                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                     #7)       (B) LOCATION: group(2,                                              #/note= "Ser or Ala"INFORMATION:                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 3                                                     #/note= "Ans or Gln"INFORMATION:                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 4                                                     #/note= "Lys or His"INFORMATION:                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                     #6, 11)   (B) LOCATION: group(5,                                              #/note= "Tyr, Trp, Phe, substitued                                                           Phe, di-s - #ubstituted Phe, homophenylalanine,                               beta-(3-pyri - #dyl)alanine, beta-(2-thienyl)alanine,                         beta-(1-naph - #thyl)alanine or beta-(2-naphthyl)alanine"      -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 8                                                     #/note= "Asn or Ala"INFORMATION:                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 9                                                     #/note= "Ile, Leu or Val"MATION:                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: Modified-sit - #e                                               (B) LOCATION: 10                                                    #/note= "His or Ala"INFORMATION:                                              -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:                                 - Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xa - #a                             #                10                                                           - (2) INFORMATION FOR SEQ ID NO:8:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 10 amino                                                          (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: circular                                              -     (ix) FEATURE:                                                                     (A) NAME/KEY: Cross-links                                                     (B) LOCATION: 1..10                                                 -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:                                 - Val Ser Asn Lys Tyr Phe Ser Asn Ile His                                     #                10                                                           - (2) INFORMATION FOR SEQ ID NO:9:                                            -      (i) SEQUENCE CHARACTERISTICS:                                          #acids    (A) LENGTH: 5 amino                                                           (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9:                                 - Val Ser Asn Lys Tyr                                                         1               5                                                             __________________________________________________________________________

We claim:
 1. A cyclic peptide compound of formula ##STR3## wherein allof X¹ through X¹¹ represent L-series amino acids; andX¹ is Val or Ala;X² is Ser or Ala; X³ is Asn or Gln; X⁴ is Lys or His; X⁵ is Tyr, Trp,Phe, substituted Phe, di-substituted Phe, homophenylalanine,β-(3-pyridyl)alanine, β-(2-thienyl)alanine, β-(1-naphthyl)alanine, orβ-(2-naphthyl)alanine; X⁶ is Tyr, Trp, Phe, substituted Phe,di-substituted Phe, homophenylalanine, β-(3-pyridyl)alanine,β-(2-thienyl)alanine, β-(1-naphthyl)alanine, or β-(2-naphthyl)alanine;X⁷ is Ser or Ala; X⁸ is Asn or Ala; X⁹ is Ile, Leu, or Val; X¹⁰ is Hisor Ala; X¹¹ is Tyr, Trp, Phe, substituted Phe, di-substituted Phe,homophenylalanine, β-(3-pyridyl)alanine, β-(2-thienyl)alanine,β-(1-naphthyl)alanine, or β-(2-naphthyl)alanine; and L is a linkingunit, such that when X¹ and X¹¹ are linked, the linear dimension betweenthe Cα carbon of amino acid X¹ and the Cα carbon of amino acid X¹¹ isbetween about 4 and 12 Ångstrom units;with the proviso that L does notcomprise two cysteines linked by a disulfide bond.
 2. The compound ofclaim 1 wherein the linear dimension between the Cα carbon of amino acidX¹ and the Cα carbon of amino acid X¹¹ is between about 5 and 10Ångstrom units.
 3. The compound of claim 1 wherein the linear dimensionbetween the Cα carbon of amino acid X¹ and the Cα carbon of amino acidX¹¹ is between about 6 and 8 Ångstrom units.
 4. The compound of claim 1wherein L is selected from the group consisting of:--CO--CH₂ --CH₂ --CH₂--S--CH₂ --CH(COOH)--NH--; --CO--CH₂ --CH₂ --CH₂ --CH₂ --S--CH₂--CH(COOH)--NH--; --CO--CH(NH₂)--CH₂ --S--CH₂ --CH₂ --CH₂ --NH--;--CO--CH₂ --CH₂ --S--CH₂ --CH₂ --CH₂ --NH--; --CO--CH₂ --CH₂ --CH₂ --CH₂--CH₂ --CH₂ --NH--; --CO--CH₂ --CH₂ --S--CH₂ --CH₂ --CH(COOH)--NH--;--CO--CH₂ --CH₂ --CH₂ --CH₂ --CH₂ --CH(COOH)--NH--; --CO--CH₂ --S--CH₂--CH(COOH)--NH--; --CO--CH₂ --S--CH₂ --CH₂ --CH(COOH)--NH--; and--CO--CH₂ -meta-phenylene-CH₂ --NH--.
 5. The compound of claim 1 havingan IC₅₀ value of less than about 10⁻⁵ molar in a competitive bindingassay to urokinase plasminogen activator receptor in vitro.
 6. Thecompound of claim 5 having an IC₅₀ value less than about 10⁻⁶ molar insaid assay.
 7. The compound of claim 6 having an IC₅₀ value less thanabout 10⁻⁷ molar in said assay.
 8. A pharmaceutical compositioncomprising(a) the cyclic peptide compound of claim 1; and (b) apharmaceutically acceptable carrier.
 9. A pharmaceutical composition,comprising:the cyclic peptide of claim 2; and a pharmaceuticallyacceptable carrier.
 10. A pharmaceutical composition, comprising:(a) thecyclic peptide of claim 3; and (b) a pharmaceutically acceptablecarrier.
 11. A pharmaceutical composition comprising:(a) the cyclicpeptide of claim 4, and (b) a pharmaceutically acceptable carrier.
 12. Apharmaceutical composition comprising:(a) the cyclic peptide compound ofclaim 5 and (b) a pharmaceutically acceptable carrier.
 13. Apharmaceutical composition comprising:(a) the cyclic peptide compound ofclaim 6; and (b) a pharmaceutically acceptable carrier.
 14. Apharmaceutical composition comprising:(a) the cyclic peptide compound ofclaim 7; and (b) a pharmaceutically acceptable carrier.
 15. Thepharmaceutical composition of claim 8 in a form suitable for injection.16. The pharmaceutical composition of claim 9 in a form suitable forinjection.
 17. The pharmaceutical composition of claim 10 in a formsuitable for injection.
 18. The pharmaceutical composition of claim 11in a form suitable for injection.
 19. The pharmaceutical composition ofclaim 12 in a form suitable for injection.
 20. The pharmaceuticalcomposition of claim 13 in a form suitable for injection.
 21. Thepharmaceutical composition of claim 14 in a form suitable for injection.22. The cyclic peptide of any of claims 2, 3, 4, 5, 6, 7, or 1, to whichis bound a detectable label.
 23. The cyclic peptide of claim 22 whereinsaid label is a radioactive, a fluorogenic, a chromogenic, or a chemicallabel.